Egress from host cells is a crucial step in the life cycle of intracellular pathogens including the parasitic protozoan Toxoplasma gondii. T. gondii can cause severe disease during pregnancy or immune dysfunction, and debilitating ocular pathology in otherwise healthy people. After intracellular replication, the parasite ruptures from host cells in a manner shared by other parasites including Plasmodium. Lytic egress frees the parasite to infect new host cells but the ensuing damage also fuels inflammation and fever, hallmarks of toxoplasmosis and malaria. We recently showed that T. gondii rapid egress and fatal acute disease both depend on the timely secretion of a pore-forming protein, TgPLP1. Our finding that TgPLP1 is released from apical micronemes exposed a novel role for these regulated secretory organelles. How TgPLP1 lyses host membranes during egress is not known. The absence of such knowledge precludes strategic efforts to extinguish its activity and alter the course of infection. Our long-term goal is to understand the roles of key microneme proteins in T. gondii infection. The objective for this funding period is to determine how TgPLP1 selectively and rapidly lyses host cell membranes during egress without causing parasite self-damage. We hypothesize that TgPLP1 pore formation is dictated by the lipid composition of its target membranes, environmental factors, and specific structural features of the protein. This assertion is based on preliminary data identifying host lipid receptors targeted for selective lysis, potential roles for pH and proteolysis in regulating pore formation, and conserved structural features of TgPLP1 predicted to drive membrane binding and oligomerization. The specific aims are: (1) Reveal the mechanism for selective cytolysis of host but not parasite membranes; (2) Uncover how TgPLP1 is active during egress but not during invasion; and (3) Identify the molecular basis for rapid pore formation. By providing a deeper mechanistic understanding of an essential step in the T. gondii life cycle, we expect the proposed work will create novel opportunities to interrupt infection and potentially ameliorate disease. PUBLIC HEALTH RELEVANCE: We seek to understand how a putative pore forming protein called TgPLP1 facilitates cell egress by the human parasite Toxoplasma gondii. TgPLP1 belongs to a family of pore forming proteins are used for both for microbial aggression and host defense. Our work will reveal mechanistic and regulatory features of pore formation that are relevant to a broad range of microbial infections and immune-related diseases.